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Proteomics, a field dedicated to studying proteins on a grand scale, has played a pivotal role in revolutionizing our comprehension of cellular processes and functions. It encompasses a diverse array of techniques aimed at characterizing proteins, including their abundance, post translational modifications (PTMs), and interactions. Among the powerful tools in the realm of proteomics, 2D electrophoresis firmly stands out as an esteemed technology for not only separating and analyzing intact proteins from complex mixtures, but also pushing the boundaries of innovation.
Sometimes referred to as 2-DE, two-dimensional gel electrophoresis serves as a potent and widely renowned method for scrutinizing protein mixtures through the use of gel electrophoresis. Although isoelectric focusing and SDS-PAGE have proven their effectiveness as individual strategies, 2D electrophoresis ingeniously combines the strengths of both techniques. By employing isoelectric focusing initially, proteins are separated and their isoelectric point (pl) is determined. Subsequently, SDS-PAGE is employed to separate these proteins once again, facilitating the identification of their molecular weight. Harnessing the power of high-throughput mass spectrometry, each protein spot on a 2D electrophoresis can be eluted and accurately identified.
As a result, 2D electrophoresis plays an instrumental role in facilitating comparisons of protein profiles between tissues, circumstances, or between samples that remain untreated and those that have undergone treatment. Its contribution resonates, thereby forging a path towards expanded knowledge and understanding of proteins in various contexts.
2D Gel Electrophoresis Service in Creative Proteomics
The comprehensive 2D electrophoresis service provided by Creative Proteomics, a leading company in the field of proteomics, continuously optimizes service workflow and utilizes the advantages of 2D-DE technology and mass spectrometry to assist scientists in better protein analysis and PTM research. With this service, complex mixtures consisting of thousands of different proteins can be separated and their relative amount can then be determined.
The service offers extreme high-resolution 2D gel electrophoresis, allowing for the separation of various protein-containing samples. With different gel sizes available, ranging from 8x7 cm to 60x30 cm, the service can accommodate diverse sample characteristics and achieve exceptional resolution. Depending on the gel size and sample properties, up to an impressive 10,000 protein spots can be resolved, providing unparalleled insights into the proteome.
Advantages of Our 2D Electrophoresis Service
High-throughput: 2D electrophoresis can accurately analyze thousands of proteins in a single run.
High resolution. This technology resolves proteins according to both pI and molecular mass, and enables the characterization of proteins with posttranslational modifications that affect their charge state.
Various computer-based tools are available: We have tools such as SameSpots, Delta2D, ImageMaster, which can be used for detection and quantification of protein spots.
Cost-efficient and affordable: While mass spectrometers represent a significant investment and require experience staff, 2D electrophoresis is relatively inexpensive.
High Flexibility: We work closely with you to design the optimal experimental scheme to meet your research objectives on budget.
2D DIGE Images for Various Sample Types
| Human Cells | Human Fluid/Tissue | Lab Animals | Bacteria & Others | |
|---|---|---|---|---|
| Jurkat cells | Breast cancer cell line | Saliva | Mouse & Rat brain | E. coli |
| PBMCs | Beta-islet | Urine | Mouse oocyte | Salmonella |
| HEK293 | Hepatocytes | Amniotic fluid | Mouse retina cells | Bacteria-secreted |
| Blood T cells | Keratinocytes | Adipose | Rat tear | Yeast |
| Blood monocyte | Platelets | Liver tissue | Drosophila | Plant |
| Dendritic cells | Blood cell exosome | Serum / Plasma | C. elegans | Rice |
| Cerebrospinal fluid | Zebrafish | Flu vaccines | ||
Protein Profiling Service by 2D Electrophoresis
Efficiently and accurately identify proteins in SDS-PAGE and 2-DE samples are critical for analyzing proteins in complex mixtures. Two-dimensional electrophoresis can separate thousands of protein spots in a single separation, making it ideal for protein profiling studies.
By comparing 2D gel images from different samples or conditions, researchers can detect differences in protein expression levels, indicating the presence or absence of specific proteins, changes in protein abundance, or the occurrence of posttranslational modifications (PTMs).
Protein profiling is a powerful tool for biomarker discovery, where differences in protein expression can be used as diagnostic or prognostic markers for diseases.
Protein profiling can be used to describe the distribution of protein isoforms and species, which can reveal details about alternative splicing and protein variants.
It enables an examination into protein quantification, which entails figuring out how much protein abundance has changed between samples or experimental groups.
Protein profiling can also be used to measure host cell protein (HCP) antibody coverage and find HCP contaminants, guaranteeing quality control in the development of biopharmaceuticals.
PTM Studies by 2D Electrophoresis
Because of its capacity to differentiate charge and size isomers of polypeptides, 2D electrophoresis is useful for researching posttranslational modifications (PTMs).
Profiling of phosphoprotein expression: Phosphorylated proteins migrate to a more acidic part of the gel, allowing researchers to investigate signaling pathways and identify phosphorylation events.
Acetylated protein expression profiling: acetylation of proteins at their amino termini or on lysine side-chains will cause them to shift to a more acidic region on the gel. Acetylation of proteins can affect protein function, interactions and subsequent or additional posttranslational modifications
Methylated protein expression profiling: Methylation alters the isoelectric point of proteins and has implications in epigenetic studies and gene regulation.
Glycosylated protein expression profiling: Glycosylation, a complex PTM, can result in changes in molecular weight and isoelectric point, making 2D electrophoresis suitable for its analysis.
Advantages of 2D DIGE Technology
A notable advancement in 2D electrophoresis is the introduction of 2D DIGE (Difference Gel Electrophoresis), which offers significant advantages over standard 2D gel electrophoresis and other proteomics techniques. One of the key advantages is its higher sensitivity. 2D DIGE employs fluorescent labeling with a sensitivity of 0.2 ng/spot, surpassing the sensitivity of standard 2D gel electrophoresis with Coomassie blue staining (100 ng/spot) or silver staining (1 ng/spot). This heightened sensitivity enables the detection of subtle changes in protein abundance, as small as 10%.
Another remarkable advantage of 2D DIGE is its higher accuracy. The technology allows for extremely high spot resolution, enabling precise spot quantitation. Even differences in protein expression as small as 10% can be reliably detected, facilitating the identification of differentially expressed proteins and providing valuable insights into biological processes.
Moreover, 2D DIGE offers exceptional reproducibility. Nearly identical data can be obtained from the same sample labeled with different fluorescent dyes on the same gel or across different gels, eliminating the need for running technical replicates. This robust reproducibility enhances the reliability of the results, ensuring consistent and trustworthy analyses.
Furthermore, 2D DIGE provides a broader spectrum of protein detection. With large 2D gel formats capable of resolving approximately 5000 protein spots, the technique enables the detection and quantitation of low-abundance proteins, large proteins, and small peptides. This wide dynamic range expands the scope of proteome analysis, facilitating the comprehensive exploration of protein profiles and posttranslational modifications.
2D Electrophoresis Workflow
The 2D electrophoresis service at Creative Proteomics follows a well-established workflow, meticulously designed to deliver high-quality results. The workflow involves several key steps:
Sample Preparation: Samples, such as purified proteins, frozen cell pellets or tissues, body fluids, cell cultures, microorganisms, plants, and more, undergo customized sample preparation protocols. This step ensures the conversion of samples to a physicochemical state suitable for 2D electrophoresis.
First-Dimension Separation by Isoelectric Focusing (IEF): In the first dimension, proteins are separated based on their isoelectric point (pI). A protein mixture is loaded at the basic end of the pH gradient gel, and under the influence of an electric field, proteins with a positive net charge migrate towards the cathode, while those with a negative net charge migrate towards the anode.
Second-Dimension Separation by SDS-PAGE: After the first-dimensional separation, the proteins are immobilized on the gel strip. The gel strip is then placed on top of an SDS-PAGE gel for the second-dimensional separation based on molecular weight. This step further resolves the proteins, allowing for precise profiling and analysis.
Visualization of Results: The separated proteins can be visualized using various staining techniques, such as blue staining or silver staining. These visualization methods enable the direct observation of protein profiles and facilitate the identification of differentially expressed proteins or PTMs.
Further Analysis: Following visualization, the protein spots of interest can be eluted and subjected to downstream analyses. Information such as molecular weight, pI, and abundance can be determined. High-throughput mass spectrometry is commonly employed for protein identification, enabling comprehensive characterization of the proteome.
Figure 1. The workflow of 2D electrophoresis.
How can I improve the resolution of my 2D gels?
To enhance resolution, consider the following strategies:
- Optimize Sample Preparation: Ensure proteins are properly extracted and purified. Avoid conditions that may lead to degradation or aggregation.
- Use IEF Strips with a Wider pH Range: Utilizing strips with a broader pH gradient can improve separation of proteins with similar pI values.
- Adjust Sample Load: Reduce the amount of sample loaded onto the gel to prevent overcrowding, which can lead to overlapping spots.
- Control Temperature: Maintain a consistent temperature during the IEF process to avoid heat-related issues that can affect protein migration.
What steps should I take if my gel cracks during electrophoresis?
Cracking in gels can significantly affect the quality of your results. To prevent and address this issue:
- Check Gel Composition: Ensure the correct concentration of acrylamide is used for your specific sample type.
- Control Polymerization Conditions: Polymerization should occur at a controlled temperature. Rapid polymerization can lead to cracks.
- Avoid Overloading the Gel: Excess sample can cause excessive pressure and lead to cracking. Optimize your loading amounts.
- Ensure Proper Casting Techniques: Make sure to avoid air bubbles during gel casting, which can create weak points in the gel.
What is the best method for staining and visualizing proteins on a 2D gel?
Staining is crucial for visualizing proteins post-electrophoresis. Here are recommended approaches:
- Coomassie Brilliant Blue Staining: This is a widely used method for general protein visualization. It is sensitive and allows for the detection of low-abundance proteins. Make sure to fix the gel before staining to enhance staining uniformity.
- Silver Staining: For higher sensitivity, silver staining is an excellent choice, especially for detecting low abundance proteins. However, it's a more complicated and time-consuming process.
- Fluorescent Stains: Consider using fluorescent stains for even higher sensitivity and quantification capabilities. Ensure you have the appropriate imaging system for detection.
How can I troubleshoot issues with protein transfer from gel to membrane?
Optimize Transfer Conditions: Check the voltage and transfer time. A common protocol uses 100 V for 1-2 hours; however, conditions may need adjustment based on gel thickness and protein size.
Check Buffer Composition: Ensure that your transfer buffer contains methanol (or isopropanol) for PVDF membranes, as this can enhance protein binding.
Use a Sandwich Method: Ensure that there's good contact between the gel and membrane without air bubbles. Consider using a transfer sandwich setup to improve contact.
Evaluate Membrane Condition: Ensure the membrane is pre-wetted appropriately. PVDF membranes should be activated in methanol, then rinsed in transfer buffer.
What should I do if bubbles form during electrophoresis?
Ensure Proper Gel Casting: When casting gels, avoid introducing air bubbles by carefully mixing and pouring the solution.
Check Electrode Connections: Poor connections can lead to arcing and bubble formation. Ensure that all connections are tight and secure.
Monitor Buffer Levels: Ensure that the buffer covers the gel completely during electrophoresis. Low buffer levels can cause the gel to dry out and form bubbles.
Use Anti-bubble Agents: Some laboratories add small amounts of surfactants to the buffer to reduce bubble formation.
What causes poor separation between protein spots, and how can I fix it?
Overloading Samples: High protein concentration can lead to overlapping spots. Reduce the sample volume.
Inconsistent pH Gradients: Check the IEF strip for proper pH gradient formation. If the gradient is not well established, consider using freshly prepared strips.
Incorrect Gel Concentration: Adjust the acrylamide concentration according to the expected size of your proteins. A higher concentration is better for smaller proteins, while lower concentrations are suited for larger ones.
What causes horizontal bands in my gel, especially when high-abundance proteins are present?
Horizontal bands can occur when high-abundance proteins aggregate, particularly if using a gel strip with the gel side facing down. To mitigate this issue, consider the following solutions:
- Use Cup Loading or Manifold Techniques: These methods involve loading samples with the gel side facing up, which helps reduce the formation of horizontal bands.
- Ensure Proper Sample Dilution: Diluting high-abundance proteins can help prevent their aggregation and the resultant horizontal bands.
Why are there horizontal bands when using narrow pH range gels?
Horizontal bands in narrow pH range gels may indicate insufficient focusing or excessive salt ions. Additional causes include:
- Detergent Concentration: Ensure the concentration of your detergent is adequate.
- Urea Concentration: Verify that urea is present in sufficient amounts to maintain protein solubility.
- DTT Concentration: Ensure DTT concentration is high enough and that the quantity used is appropriate.
- Sample Loading Position: Improper loading position, such as using cup loading, can affect band formation.
- Protease Activity: Check for active proteases in your sample that may degrade proteins.
- Gel Swelling Time: Insufficient swelling time of the gel strips can lead to uneven protein migration.
- Presence of Insoluble Proteins: Centrifuge samples at 40,000 g for 1 hour to remove any insoluble proteins.
- CO2 in the Air: High levels of CO2 can affect pH and cause issues during focusing.
How does reagent freshness impact my results?
Using stale reagents can significantly affect your results. For instance, TEMED, while used in small quantities, should be replaced annually to ensure optimal performance.
What impact does reagent quality have on protein spot imaging?
The quality of Tris used in buffers can influence the outcome of your electrophoresis. Poor-quality Tris may lead to inconsistent pH and buffer conditions, ultimately affecting protein separation and visualization.
What steps can I take if proteins are lost during transfer?
Improper transfer conditions can result in protein loss. Ensure:
Voltage Settings: Maintain appropriate voltage settings, especially during the initial phase. For an 18 cm gel, avoid exceeding 2 W/gel during the first 45 minutes to prevent excessive heat and protein loss.
What should I consider when preparing plant samples for electrophoresis?
Plant samples often contain polysaccharides that can interfere with the electrophoresis process. To ensure cleaner samples:
Clean-up Treatment: Use clean-up procedures to remove polysaccharides and other contaminants before running the electrophoresis.
Why are there vertical bands in my protein spots?
Vertical bands can result from inadequate DTT during the equilibration phase, affecting the reduction of disulfide bonds. Ensure that DTT is present in sufficient concentrations during this process to minimize vertical banding.
What causes high salt ions in my samples, and how can I resolve this?
Excessive salt ions can lead to failure in isoelectric focusing. To prevent this issue:
De-salting Techniques: Remove excess salts by thoroughly washing cells. After centrifugation, ensure all PBS is removed, or use a washing buffer containing 250 mM sucrose and 10 mM Tris to cleanse the cells effectively.
How should I handle loading alkaline pH range gels?
For gels with a pH range of 6-11, it is recommended to use cup loading for improved sample application, especially for alkaline pH range gels.
What issues arise from storing second-dimension gels at low temperatures?
Storing second-dimension gels at excessively low temperatures can cause SDS precipitation, affecting gel integrity. To avoid this:
Optimal Storage Conditions: Store gels at 4–8°C for short periods (2 days to 2 weeks). Before running the second dimension, remove the gel from cold storage and allow it to acclimate to room temperature to prevent precipitation issues.
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